Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl’s ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls’ ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.

x是了解如何实现复杂身体部位再生的强大模型，而哺乳动物的这种能力受到严重限制。可以在哺乳动物中检查促进轴突再生的因子，以确定它们在这种情况下是否表现出改变的活性。此外，禁止轴突再生的因素可以提供对再生能力不强的物种中存在的机制的关键见解。我们试图确定我们是否可以在实验上损害compromise的再生四肢的能力，如果可以，则发现可能导致其无法再生的分子变化。我们发现，反复进行肢体截肢严重损害了x的引发肢体再生的能力。使用RNA-seq，我们观察到大多数差异表达的转录本在反复截肢受损的四肢中被过度激活，表明这些基因的错误调节会拮抗再生。为了证实我们的发现，我们另外分析了两性调节蛋白，一种类似于EGF的配体，在受感染的动物中异常上调。在正常的肢体再生过程中，早期伤口表皮表达双调蛋白，错误表达该因子会导致伤口上皮增厚，再生开始延迟和严重的再生缺陷。总体而言，我们的结果表明，反复截肢的肢体可能会经历持续的伤口愈合反应，这会干扰其启动再生程序的能力。这些发现对人类再生医学具有重要意义。